As Alzheimer's disease pathogenesis is associated with the formation of insoluble aggregates of amyloid beta-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices. Quantitative analysis revealed increasing fluorescence signal intensity with increasing plaque load of the animals, and significant binding of AOI987 was observed for APP23 transgenic mice aged 9 months and older. Thus, AOI987 is an attractive probe to noninvasively monitor disease progression in animal models of Alzheimer disease and to evaluate effects of potential Alzheimer disease drugs on the plaque load.
No abstract
Infrared attenuated total reflection (IR-ATR) spectroscopy was used to study conformational and topological aspects of the interaction between two adrenocorticotropin fragments and dioleoylphosphatidylcholine membranes. Corticotropin-(1-10)-decapeptide, ACTH1-10, was found to exist as a rigid antiparallel pleated sheet structure in dry membranes. In aqueous environment, it completely escaped from the lipid. This dominant preference for the aqueous phase is a possible explanation for the very low biological potency of ACTH1-10 in some assays. On the other hand, the very potent corticotropin-(1-24)-tetracosapeptide, ACTH1-24, was firmly incorporated into dry and wet membranes. Aqueous environment even promoted the peptide-lipid interaction. Under these latter conditions, part of the molecule entered the bilayer and adopted a helical structure with the axis oriented perpendicularly to the bilayer plane. Contact of a 0.1 mM solution of ACTH1-24 in liquid deuterium oxide with the pure lipid membrane system resulted in measurable adsorption of the peptide to the membrane with the same conformational and topological characteristics as described above (perpendicularly oriented helix entering the bilayer). The helical part of the ACTH1-24 molecule entering the bilayer was the quite hydrophobic N-terminal decapeptide unit ("message" segment). The adjacent hydrophilic C-terminal tetradecapeptide unit ("address" segment) remained on the membrane surface. As the message region is essential for triggering corticotropin receptors, its intrusion into the membrane and its adoption of an oriented, helical conformation may facilitate receptor stimulation.
Circular dichroism (CD) spectra of C-terminal deletion mutants of the HIV-1 Rev protein, Rev M9 delta 14 (missing aa 68-112) and Rev M11 delta 14 (lacking aa 92-112), indicated that Rev contains 46-49 residues in alpha-helical conformation within the N-terminal 71 or 95 amino acids of the 116 residue protein. Complexation with a 40-nucleotide fragment of the Rev responsive element, RRE, (G39 to C78), containing the minimal element for Rev binding, induced an A to B form structural transition in the RRE fragment, whereas the percentage of alpha-helical conformation in the protein stays constant on substrate binding. When complexed to the RNA, neither mutant protein showed structural changes upon raising the temperature to 40 degrees C, as determined by the lack of decrease of the signal intensity at 222 nm, indicative for alpha-helical conformation. In contrast, Rev M9 delta 14, which is shorter than Rev M11 delta 14 by 24 amino acids, in the absence of RNA, lost about 60% of the spectral minima at 222 nm at the same temperature. The Rev M11 delta 14 mutant, in the absence of RNA, showed a decrease of 20% in spectral intensity upon heating to 40 degrees C. Free and RNA-bound mutant proteins showed reversible transitions upon heating to 80 degrees C and subsequent cooling down to 10 degrees C overnight. The Rev peptide Cys 75-93, spanning the Rev transactivation domain, showed secondary structure in 40% and 60% hexafluoropropanol (HFP) solutions.(ABSTRACT TRUNCATED AT 250 WORDS)
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